22 DNA Breakage Detection-FISH (DBD-FISH)
285
4. The slide is left at 4°C for 5 min to allow gel solidification.
5. Half of the slide is irradiated with a Philips RT-100 machine, while kept
on a cold plate to avoid DNA repair. The other half (protected or out of
the irradiation field) serves as a control with undamaged cells.
Note: Cells may be damaged by physical or chemical agents either in
the gel or while in a monolayer or suspension. We prefer to produce
DNA breakage in cells embedded in microgels, avoiding possible mechanical DNA breaks by manipulation, or repairing when mixing with
agarose at 37°C.
6. Immediately, the coverslip is removed by pulling carefully from an
overhanging end. Immersion in abundant cold (4°C) isotonic saline
solution, without buffer, for 5-10 min, aids coverslip removal.
7. The slide is horizontally immersed in abundant alkaline unwinding
solution, freshly prepared, at 22°C for 2-5 min. Time, temperature
and NaCI or NaOH concentration strongly influence the amount of
ssDNA generated (Rydberg 1975). Alkali-labile sites are also transformed into ssDNA by this solution.
8. The slide is transferred horizontally to abundant neutralising and lysing solution 1 for 20 min and then to abundant neutralising and lysing
solution 2 for 10 min, both at room temperature. 0.4 M Tris, pH 7.5,
neutralises the alkali and stops DNA denaturation, and DTT breaks
protein disulphide bonds thereby allowing their extensive removal
by detergent and highly concentrated salt solutions. Though unwinding solution partially lyses cells, specific lysing steps improve the accessibility of DNA probes to ssDNA.
Note: Once the alkali is neutralised, DNA breaks produced afterwards,
e.g. by manipulation, are irrelevant since they are not transformed into
ssDNA, and so will not be detected by the probes.
9. The slide is left horizontally in abundant TBE buffer for 15 min, at
room temperature.
10. After immersion in 70%, 90% and 100% ethanol baths, 2 min each, at
room temperature, the slide is air-dried by incubation at 37°C for 15-30
min. The agarose film turns into averythin film that is well-attached to the
slide. If necessary, the slides can be stored dry, in the darkness, at room
temperature, for a week (and probably more), before hybridisation.
DNA damage
induction
Transformation of
DNA breaks into
ssDNA
NeutraJising and
protein removal
Washing and
dehydration
285
4. The slide is left at 4°C for 5 min to allow gel solidification.
5. Half of the slide is irradiated with a Philips RT-100 machine, while kept
on a cold plate to avoid DNA repair. The other half (protected or out of
the irradiation field) serves as a control with undamaged cells.
Note: Cells may be damaged by physical or chemical agents either in
the gel or while in a monolayer or suspension. We prefer to produce
DNA breakage in cells embedded in microgels, avoiding possible mechanical DNA breaks by manipulation, or repairing when mixing with
agarose at 37°C.
6. Immediately, the coverslip is removed by pulling carefully from an
overhanging end. Immersion in abundant cold (4°C) isotonic saline
solution, without buffer, for 5-10 min, aids coverslip removal.
7. The slide is horizontally immersed in abundant alkaline unwinding
solution, freshly prepared, at 22°C for 2-5 min. Time, temperature
and NaCI or NaOH concentration strongly influence the amount of
ssDNA generated (Rydberg 1975). Alkali-labile sites are also transformed into ssDNA by this solution.
8. The slide is transferred horizontally to abundant neutralising and lysing solution 1 for 20 min and then to abundant neutralising and lysing
solution 2 for 10 min, both at room temperature. 0.4 M Tris, pH 7.5,
neutralises the alkali and stops DNA denaturation, and DTT breaks
protein disulphide bonds thereby allowing their extensive removal
by detergent and highly concentrated salt solutions. Though unwinding solution partially lyses cells, specific lysing steps improve the accessibility of DNA probes to ssDNA.
Note: Once the alkali is neutralised, DNA breaks produced afterwards,
e.g. by manipulation, are irrelevant since they are not transformed into
ssDNA, and so will not be detected by the probes.
9. The slide is left horizontally in abundant TBE buffer for 15 min, at
room temperature.
10. After immersion in 70%, 90% and 100% ethanol baths, 2 min each, at
room temperature, the slide is air-dried by incubation at 37°C for 15-30
min. The agarose film turns into averythin film that is well-attached to the
slide. If necessary, the slides can be stored dry, in the darkness, at room
temperature, for a week (and probably more), before hybridisation.
DNA damage
induction
Transformation of
DNA breaks into
ssDNA
NeutraJising and
protein removal
Washing and
dehydration